Thrombin is an important serine proteinase component of the blood coagulation cascade. Besides initiating blood clotting by cleaving fibrinogen, thrombin activates other hemocoagulant factors including factors V, VIII and XIII and the anticoagulant enzyme protein C. Thrombin is also a potent platelet activator that impairs thrombolysis mediated by tissue plasminogen activator in vivo. Thus, thrombin's positive feedback regulation serves to amplify hemostatic events but causes life-threatening thrombi in response to aberrations with vascular and cerebrovascular arteries.
Given the diverse functions of this enzyme, its inhibition by potent and specific compounds could provide an invaluable addition to the treatment of disorders related to thrombosis. These include: coronary artery disease, cerebrovascular disease, peripheral arterial occlusive disease, deep vein thrombosis, and pulmonary embolism.
The most potent inhibitor of thrombin known is hirudin, a family of iso-proteins isolated from the glandular secretions of the leech Hirudo medicinalis. The anticoagulant properties of hirudin have been known for a long time. However, it has so far been of little therapeutic value since the formulation of this protein in a readily efficient and administrable form seems to be difficult as both enteral and cutaneous absorptions are very low so it has not been possible to produce adequate levels of the protein in the bloodstream.
Furthermore, clinical use of hirudin isolated from leech extracts is unlikely because of its limited quantity, expense, and allergic reactions which may commonly occur upon administration of foreign proteins having the size of hirudin.
In his publication entitled "Pharmacology of selective thrombin inhibitors", (1988), Nouv. Rev. Fr. Hematol., 30, pages 161-165, Markwardt provided further clinical information on hirudin based on results of pharmacological studies performed for both natural and synthetic thrombin inhibitors.
The author makes general observations concerning hirudin, mentioning that the peptide, which contains a highly acidic C-terminal portion, is highly specific for .alpha.-thrombin. He then concludes that the C-terminal portion of hirudin is likely to bind to the anionic binding site region of the enzyme whereas the compact N-terminal portion appears to bind to the active site region of the enzyme.
It has been found that native desulfo hirudin.sup.45-65 inhibits fibrinogen clotting by both bovine and human .alpha.-thrombin in a dose dependent manner. The IC.sub.50 value of 940.+-.200 nM for bovine .alpha.-thrombin is in good agreement with the reported value of plasma fibrin clot formation by the same fragment and three times lower than hirudin.sup.55-65, which had been assigned as the minimum core required for anticoagulant activity. It has also been demonstrated that the same peptides were consistently more potent against human .alpha.-thrombin than bovine .alpha.-thrombin.
Various prior art documents have also demonstrated that the active fragment of the amino acid sequence of hirudin appears to be the amino acid sequence including amino acids 45 to 65. Hence, efforts have been made to enhance the inhibitory activities of the peptide by substituting some of the amino acids present in this sequence.
Krstenansky et al., in "Antithrombin properties of C-terminus of hirudin using synthetic unsulfated N.alpha.-acetyl-hirudin", (1987), Febs Letters, Vol. 211, No. 1, pages 10-16, describe the synthesis of the C-terminal fragment unsulfated N.alpha.-acetyl-hirudin.sup.45-65. The authors refer to previous work (Chang, J.-V., FEBS Letters, 164, 307 (1983)) and mention that this fragment could potentially contain two specific binding domains, one binding to the catalytic site of thrombin and the other binding to another recognition site on thrombin. This was concluded not to be the case by either authors.
Still, the authors demonstrated that the 45-65 sequence of hirudin has the ability to inhibit clotting activity as well as the release of fibrinopeptide A by thrombin. They also suggested that the same sequence of hirudin.sup.45-65 may not be directly involved in the binding with the catalytic site of thrombin since the amidolytic properties of thrombin toward synthetic substrates is not perturbed.
In the Krstenansky et al. article entitled "Anticoagulant peptides: nature of the interaction of the C-terminal region of hirudin with a noncatalytic site on thrombin", (1987), J. Med. Chem., 30, pages 1688-1691, the authors report that the minimum active sequence at the noncatalytic binding site of thrombin is hirudin.sup.56-64. Based on this assumption, the authors report the synthesis of several C-terminal hirudin.sup.54-65 analogs and their ability to inhibit thrombin-induced fibrin clot formation for the purpose of establishing the nature of the interaction between hirudin.sup.56-64 and a noncatalytic binding site of thrombin.
In their conclusion, the authors mention that the C-terminal region of hirudin may bind to a region of fibrinogen binding on thrombin that is not the region proposed so far in the literature.
In the articles by Dodt et al. (Interaction of site specific hirudin variants with .alpha.-thrombin, (1988), Febs Letters, Vol. 229, No. 1, pages 87-90), Degryse et al. (Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin, (1989), Protein Engineering, Vol. 2, No. 6, pages 459-465) and Braun et al. (Use of site-directed mutagenesis to investigate the basis for the specificity of hirudin, (1988), Biochemistry, 27, pages 6517-6522), the authors report the results of site-directed mutagenesis performed on the hirudin gene. The inhibition of thrombin by mutant hirudin peptides is studied.
In these publications, the authors studied mutations effected on the whole protein and did not restrict themselves to the 45-65 segment of hirudin. Furthermore, the modifications performed on the 45-65 segment were restricted to a single modification, usually at position 47, to illustrate that this residue does not interact with the active site, although these publications also show mutations at positions 51, 57, 58, 61 and 62.
In a similar fashion, the article by Dodt et al. entitled "Distinct binding sites of Ala.sup.48 -Hirudin.sup.1-47 and Ala.sup.48 -Hirudin.sup.48-65 on .alpha.-thrombin", (1990), The Journal of Biological Chemistry, Vol. 265, No. 2, pp. 713-718, describes experiments aimed at conducting site-directed mutagenesis of hirudin at position 48 in the sequence. The work done by Dodt et al. in this case seems to have been restricted to the substitution of alanine for proline at this position in order to facilitate the required proteolysis necessary for their experiment.
Finally, Maraganore et al., in "Anticoagulant activity of synthetic hirudin peptides", (1989), The Journal of Biological Chemistry, Vol. 264, No. 15, pages 8692-8698, Dennis et al. in "Use of fragments of hirudin to investigate thrombin-hirudin interaction", (1990), Eur. J. Biochem. 188, pages 61-66 and Chang et al. in "The structural elements of hirudin which bind to the fibrinogen recognition site of thrombin are exclusively located within its acidic C-terminal tail", (1990), Febs., Vol. 261, No. 2, pages 287-290, describe the synthesis and anticoagulant properties of a number of peptides whose sequences are based on the sequence of various fragments of native hirudin.
Compounds having anticoagulating properties are valuable therapeutics which may be used in vivo in the treatment of various pathologic states. Among the most important conditions in which an anticoagulant treatment may be useful, there may be mentioned myocardial infarction, pulmonary embolism and cerebral vascular diseases, deep vein thrombosis and other indications of thrombotic disorders.
Currently available anticoagulants are in many respects unsatisfactory. For example, heparin has been employed to inhibit the activity of thrombin and therefore in the treatment of conditions such as venous thrombosis and thrombo embolism. However, heparin exhibits a wide array of undesirable side effects that demonstrate the need for anticoagulants presenting more favorable toxicity levels.
The design of low molecular weight and specific inhibitors of thrombin that utilize accessory binding loci remote from or in conjunction with the catalytic center, similar to the way fibrinogen or hirudin binds to thrombin, constitutes a challenge in protein chemistry. Conceivably, such a multifunctional inhibitor integrates two or more recognitive elements, separated by a suitable spacer, that favor multiple simultaneous interactions and which could manifest enhanced potency and specificity. Incorporation of "foreign" chemical elements embodied in a structure of low molecular weight could confer resistance against proteolysis and favourable bioavailability. Also, because they are smaller than hirudin, these compounds are less likely to stimulate an undesirable immune response in patients treated with them.
PCT application WO 91/02750 indicates that certain thrombin inhibitors possess catalytic site directed moieties that may be cleaved slowly or not cleaved at all. However, all they disclose are modified bonds between the Arg and Gly or Pro such as Arg[psi CH.sub.2 --NH]-Gly; .beta.-HomoArg-Gly; .beta.-HomoArg-Pro; .beta.-HomoArg-Val; or Arg-[.psi.CO--CH.sub.2 ]--CH.sub.2 --(CONH)-Gly. There is no indication that the Gly or Pro amino acid may be completely eliminated and followed by a synthetic linker that is completely resistant to thrombin cleavage.